The emergence of pathogens that cause increased morbidity and mortality is a major public health concern. Clostridium difficile is a major cause of hospital acquired infection and is the leading cause of antibiotic-associated diarrhea. In the past decade, two epidemic ribotypes (RT027 and RT078) of C. difficile have emerged as major ribotypes in hospital outbreaks around the world despite not being observed at significant levels prior to the year 2000. These ribotypes are associated with increased morbidity and mortality and have been classified as hypervirulent. However, the mechanistic basis for why these strains began to emerge in the early 2000s is unclear. We have found that RT027 and RT078 strains have acquired the ability to grow on the disaccharide trehalose more efficiently than other ribotypes of C. difficile. Our preliminary data shows that the mechanistic bases for the improved growth in these two ribotypes are genetically and mechanistically distinct, suggesting convergent evolution of improved trehalose metabolism in these phylogenetically distinct ribotypes. In addition, we have found that disrupting the ability of a RT027 strain to utilize trehalose dramatically attenuates disease severity, suggesting that trehalose metabolism contributes to the hypervirulent nature of RT027 and RT078 strains. Trehalose, a disaccharide of glucose, became a widely used food supplement after being granted GRAS status approval by the FDA in 2000 and EFSA in Europe in 2001. Trehalose has a number of desirable properties as a sugar additive due to the alpha, alpha 1-1 linkage that is heat and acid stable, which renders it 45% less sweet than sucrose. Normally found in foods such as mushrooms and shellfish, trehalose is now added to numerous food and drink products ranging from ground beef to ice cream. The addition of trehalose to the worldwide food supply coincides with the first reports of RT027 and RT078 strains in the early 2000s, even though these ribotypes were present in hospitals as far back as 1985. We propose that addition of increased levels of trehalose to the food supply has selected for RT027 and RT078 strains due to their ability to more efficiently metabolize trehalose and has contributed to hospital epidemics. We will use two recently developed models in our laboratory, human fecal minibioreactor array model of C. difficile invasion and a humanized microbiota mouse model of C. difficile infection, to study the impact of trehalose and improved trehalose metabolism on the ability of RT027 and RT078 strains to cause C. difficile infection. We will also investigate the mechanistic bases for improved trehalose metabolism of RT027 and RT078 strains. Finally, we will assess what impact improved trehalose metabolism has on colonization dynamics, disease severity and carriage in animals of these hypervirulent C. difficile ribotypes. The following specific aims are proposed: Aim 1. Investigate the mechanism of how trehalose metabolism by C. difficile increases disease severity. Aim 2. Understand the mechanistic basis for improved trehalose metabolism in RT027 C. difficile. Aim 3. Understand the role of the acquired trehalose operon on RT078 fitness and animal carriage. We expect this work will establish a link between the recent addition of trehalose in the human diet and the emergence of epidemic C. difficile ribotypes. This will provide the first direct evidence of the alteration of the human diet directly impacting the pathogenesis of an enteric infection.